Anaerobic degradation of steroid hormones by novel denitrifying

122 Pages·2006·1.95 MB·English

Anaerobic degradation of steroid hormones
by novel denitrifying bacteria

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Biologe

Michael Fahrbach

aus Bad Mergentheim (Baden-Württemberg)

Berichter: Professor Dr. Juliane Hollender
Professor Dr. Andreas Schäffer

Tag der mündlichen Prüfung: 12. Dezember 2006

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Table of Contents

1 Introduction.....................................................................................................................1
1.1 General information on steroids...............................................................................1
1.2 Steroid hormones in the environment.......................................................................2
1.2.1 Natural and anthropogenic sources and deposits............................................2
1.2.2 Potential impact on the environment................................................................3
1.2.3 Fate of steroid hormones..................................................................................4
1.3 Microbial degradation of steroid hormones and sterols............................................5
1.3.1 Aerobic degradation..........................................................................................5
1.3.2 Anaerobic degradation......................................................................................7
1.4 Aim of dissertation....................................................................................................9
2 Materials and Methods.................................................................................................11
2.1 Microbiological methods.........................................................................................11
2.1.1 Sources of bacteria.........................................................................................11
2.1.2 Media and growth conditions..........................................................................11
2.1.3 Anoxic cultivation techniques..........................................................................13
2.1.4 Isolation, purity control and maintenance.......................................................14
2.1.5 Characterization of isolated bacteria..............................................................14
2.1.6 Phylogenetic analysis.....................................................................................15
2.1.7 MPN dilution series.........................................................................................15
2.1.8 Quantification of steroid hormone oxidation and denitrification......................16
2.1.9 Degradation time course.................................................................................16
2.2 Biochemical methods .............................................................................................17
2.2.1 Preparation of cell-free extracts for proteomics..............................................17
2.2.2 Two-dimensional gel electrophoresis.............................................................18
2.2.3 In-gel digestion...............................................................................................19
2.3 Analytical methods.................................................................................................19
2.3.1 Analysis of nitrite and nitrate by ion chromatography.....................................19
2.3.2 Analysis of dinitrogen monoxide and dinitrogen by gas chromatography.......20
2.3.3 Analysis of steroid hormones by HPLC..........................................................21
2.3.4 Proteomic analysis..........................................................................................21
2.3.5 Protein determination......................................................................................22
2.3.6 Chemotaxonomy.............................................................................................23
2.4 Chemicals...............................................................................................................23
3 Results...........................................................................................................................25
3.1 Characterization of strain AcBE2-1T.......................................................................25
3.1.1 Enrichment and isolation................................................................................25
Table of Contents

3.1.2 Physiological, cytological, and morphological properties................................26
3.1.3 Environmental significance.............................................................................28
3.1.4 Quantification of steroid hormone degradation and denitrification..................29
3.1.5 Phylogenetic analysis and chemotaxonomy...................................................30
3.2 Proteomic analysis of strain AcBE2-1T...................................................................32
3.3 Characterization of strain FST.................................................................................40
3.3.1 Enrichment and isolation................................................................................40
3.3.2 Physiological, cytological, and morphological properties................................41
3.3.3 Quantification of steroid hormone degradation and denitrification..................43
3.3.4 Phylogenetic analysis and chemotaxonomy...................................................46
4 Discussion....................................................................................................................49
4.1 Description of strain AcBE2-1T as Denitratisoma oestradiolicum gen. nov., sp. nov.
……………………………………………………………………………………………..49
4.1.1 Physiology......................................................................................................49
4.1.2 Taxonomy.......................................................................................................50
4.2 Proteomic analysis of Denitratisoma oestradiolicum AcBE2-1T..............................53
4.3 Description of strain FST as Steroidobacter denitrificans gen. nov., sp. nov..........60
4.3.1 Physiology......................................................................................................60
4.3.2 Taxonomy.......................................................................................................63
4.4 General discussion.................................................................................................67
5 Summary.......................................................................................................................71
6 Zusammenfassung.......................................................................................................73
7 References....................................................................................................................75
8 Appendix.......................................................................................................................87
8.1 Phylogenetic analysis of Denitratisoma oestradiolicum..........................................87
8.2 Assimilation equations of estradiol and testosterone..............................................88
8.3 Quantification of steroid hormone degradation and denitrification..........................89
8.4 Complete datasets of time course experiments......................................................90
8.5 Enrichment cultures................................................................................................97
8.6 Proteomic analysis of Denitratisoma oestradiolicum AcBE2-1T..............................98
8.7 Table of Figures....................................................................................................111
8.8 List of Tables........................................................................................................114
9 Acknowledgements....................................................................................................115
List of Abbreviations

% (v/v) % volume per volume
% (w/v) % weight per volume
ARB Software for sequence data analysis
ATCC American Type Culture Collection
BLAST Basic Local Alignment Search Tool
Casein-peptone soymeal-peptone agar. The medium is identical
CASO agar
with trypticase soy agar (TSA)
CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate
Da Dalton (molecular weight)
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
DNase I Endonuclease that nonspecifically cleaves DNA
Deutsche Sammlung von Mikroorganismen
DSMZ
und Zellkulturen GmbH
DTT Dithiotreithol
E1 Estrone
E2 Estradiol
EE2 Ethinyl estradiol
ESI Electrospray ionization
FAD Flavin-adenine dinucleotide
FISH Fluorescence in situ hybridization
G+C content of the DNA Guanine+Cytosine content of the DNA
GC Gas chromatography
HEPES N-[2-hydroxyethyl] piperazine-N’-[ethansulfonic acid]
HPLC High performance liquid chromatography
IEF Isoelectric focussing
IMG system Integrated Microbial Genomes system
IPG strip Immobilized pH gradient strip
JCM Japan Collection of Microorganisms
Log K Log octanol-water partition coefficient
OW
MPN Most probable number
MS Mass spectrometry
MS/MS Tandem mass spectrometry
NA No data available
NAD(P) Nicotinamide-adenine dinucleotide (phosphate)
ND Not determined
PCR Polymerase chain reaction
pI Isoelectric point
R2A agar A low-nutrient solid medium
RNA Ribonucleic acid
RNase Endonuclease that nonspecifically cleaves RNA
rRNA Ribosomal RNA
SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis
SL Trace element solution
T2 Testosterone
TCA Trichloroacetic acid
TGS buffer Tris-glycine-sodium dodecylsulfate buffer
TLC Thin layer chromatography
Tris Tris-[hydroxymethyl]-aminomethane

Introduction
1 Introduction
1.1 General information on steroids
Steroids are isoprenoic compounds that are of great importance in biology, medicine, and
chemistry and that are found in multiple forms in the environment (Fig. 1). One class
comprises the sterols with cholesterol 5 as the main representative in animals required to
build and maintain the cell membranes. Plant sterols (phytosterols) like β-sitosterol (not
shown) also act as structural components in herbal cell membranes. Another class are the
steroid hormones derived biosynthetically from cholesterol, including the estrogens (estradiol
1 and estrone 2) and the androgens (testosterone 3 and 4-androstene-3,17-dione 4). These
signal compounds are regulating metabolism, growth and reproduction in vertebrates.
Estradiol, formed by the ovary and placenta, is the most potent naturally occurring steroid
hormone in female organisms, whereas testosterone is the most important one in male
organisms, formed by the testis (Koolman et al., 1998).

OH OH
18
12 17
11 16
C 13 D 19
1 9
10
2 14 15
A B 8
HO 3 5 7 O HO
4 6 1 3 5
OH
O O
C C
HO O HO
2 4 6

Fig. 1. Molecular structures of estradiol 1, estrone 2, testosterone 3, 4-androstene-3,17-dione 4,
cholesterol 5, and ethinyl estradiol 6. The nomenclature of the carbon atoms and the different rings is
shown.

Like their biosynthetic precursor cholesterol, the four rings of the steroid skeleton of
estrogens and androgens are in trans-trans-trans conformation (Breitmaier & Jung, 1995).
One main difference between cholesterol and steroid hormones is the absence of the
1
Introduction

aliphatic side chain (Fig. 1). The aromatic ring A in estradiol shows phenolic properties.
Oxygen-dependent aromatization of testosterone to estradiol results in the removal of the
methyl group 19 between ring A and B (Stryer, 1996). Steroid hormones of lower estrogenic
activity are estrone and 4-androstene-3,17-dione. Ethinyl estradiol 6 is a synthetic derivative
of estradiol and is the key component of oral contraceptives. The acetylene residue at
position 17 circumvents an oxidation at this carbon atom and makes this compound more
recalcitrant in the environment. Basically, steroids share similar chemical characteristics
(Table 1), as they display low water solubility and comparatively high melting points. All these
molecules possess one or two quaternary carbon atoms, C-10 and C-13.

Table 1. Selected physicochemical properties of steroidal hormones
Steroid homone Molecular Water Log K Melting References
OW
weight solubility point [°C]
[g/mol] [mg l-1]
17β-Estradiol 272.4 3.9-13.3 3.1-4.0 171 Hanselman et al., 2003
Estrone 270.4 0.8-12.4 3.1-3.4 259 Hanselman et al., 2003
17α-Ethinyl estradiol 296.4 32.0 3.6-4.1 183 Lee et al., 2003
Testosterone 288.4 18.0-25.0 3.2 155 Lee et al., 2003
4-Androstene-3,17-dione 286.4 37.0-41.0 NA* 173 Lee et al., 2003
Cholesterol 386.6 2.0 NA* 149 Windholz et al., 1983

* No data available.

1.2 Steroid hormones in the environment
1.2.1 Natural and anthropogenic sources and deposits
Estradiol and estrone that were detected in the aquatic environment mainly originate from
municipal effluent discharge (Ternes et al., 1999b), runoff from agricultural production, and
farmyard manure applied as organic fertilizer (Hanselman et al., 2003). The importance of
estrogens from animal sources has to date been of less concern than their discharge from
wastewater treatment plants, but runoff from manure largely contributes to the entry in the
environment (Hanselman et al., 2003; Raman et al., 2004). Cattle and poultry manure have
also been reported as a source of the environmental loadings of testosterone (Lee et al.,
2003). It is clear that intensive animal breeding could generate large quantities of both the
steroidal estrogens and androgens from urinary and fecal deposition and indeed high
concentrations (up to 2 µg l-1) of estradiol and testosterone have been found in runoff from
poultry manure (Finlay-Moore et al., 2000). Further significant sources of androgens appear
2
Introduction
to include pulp and paper mill effluents and sewage treatment effluents (Jacobsen et al.,
2005). Another origin could be the microbial conversion of phytosteroidal compounds (e.g.,
β-sitosterol) to steroid hormones including 4-androstene-3,17-dione in anoxic aquatic
sediments (Jenkins et al., 2004).
In addition, steroids can be found as fossil fuel markers in coals, petroleum and sedimentary
rocks. These markers represent modified molecules of biochemical precursors like
cholesterol and other steroids, formed by microbial degradation, pressure, temperature and
mineral catalysis, while being buried for millions of years in deep sediments. In most cases
these steroids are thermodynamically more stable than the steroids found in the organisms
but possess the basic structure of their predecessors. They can be used to determine the
constitution of a community of organisms in a specific time of earth history, or to trace
contamination of soils, plants and groundwater by petrol and petroleum derived products
(Mackenzie et al., 1982; Payet et al., 1999).

1.2.2 Potential impact on the environment
Steroid hormones are frequently detected in the environment and are likely to cause
endocrine disrupting effects in aquatic wildlife (Sumpter & Johnson, 2005; Hanselman et al.,
2003) at concentrations in the nanogram per liter range. Endocrine disrupting chemicals
(EDCs) have been defined as “exogenous agents that interfere with the production, release,
transport, metabolism, binding, action, or elimination of the substance in the body of an
organism responsible for the maintenance of homeostasis and the regulation of
developmental processes” (Kavlock, 1991). The potential endocrine effects of estrogens,
such as vitellogenin production and feminization of male fish, have been well documented
(Panter et al., 1998; Jobling et al., 1998). Although the study of estrogens has received
considerable attention, much less effort has been directed at studying the potentional
endocrine-disrupting effects of androgens such as testosterone. Information of androgens in
the environment is limited, but aquatic organisms downstream of pulp and paper mills have
demonstrated biological responses consistent with exposure to these substances, including
masculinization of female fish (Howell & Denton, 1989; Thomas et al., 2002).
Besides their potential impact on internal physiological signal pathways, steroid hormones
also do seem to have a disrupting effect on chemical signalling pathways between different
organisms. These external signalling pathways include such fundamental processes like the
nodulation in leguminous roots mediated by phytoestrogens (Fox, 2004). Phytoestrogens like
the flavonoid luteolin act as recruiting signals to attract soil bacteria of the genus
Sinorhizobium, responsible for the symbiotic nitrogen fixation. Recent investigations have
shown that many of the same synthetic and natural chemicals that disrupt endocrine
3
Introduction

signalling in vertebrates also disrupt the binding of phytoestrogens to the bacterial nodulation
D protein (NodD) receptor. As a result the potential binding of estrogens to the bacterial
NodD receptor could have a destabilizing influence on the establishment of this vital
symbiotic relationship.

1.2.3 Fate of steroid hormones
Concern over the potential negative ecological effects of steroid hormones from human- and
animal-derived wastes has resulted in an increased interest regarding the mobility and
perstistence of these compounds in the environment. Removal of steroid hormones from
water, sediments, and soils is expected to be largely the result of a combination of sorption
and biodegradation. Being predominantly hydrophobic organic compounds of low volatility,
sorption to the solid phase is likely to be a significant process. Studies by Holthaus et al.
(2002) about the sorption of estradiol to river sediments revealed that less than 1 % of the
present steroid are predicted to be removed from the aqueous phase by suspended
sediments. Andersen et al. (2003) reported, that sorption of estradiol and estrone to activated
sludge and anoxic sewage sludge occurred to a minor degree and the sorbed steroids
appear to decrease slightly along the treatment train. The strong decrease of dissolved
estrogens along the treatment train despite the almost equal amounts of sorbed estradiol and
estrone in the activated sludge of the same sample indicates slow sorption kinetics and no
equilibrium between the sorbed and dissolved estrogens. Soils were also found to bind
estrogens (Hanselman et al., 2003; Lee et al., 2003). In addition, androgens have been
shown to be sorbed to soils or to be accumulated in sediments (Lee et al., 2003;
Jenkins et al., 2003).
Steroid hormones are mainly excreted from humans and livestock as soluble conjugates
(e.g. sulfate- and glucuronic acid esters), which are cleaved during wastewater treatment
(Ternes et al., 1999a). Recent studies demonstrated that unconjugated, active estrogens are
degraded under oxic conditions during normal activated sludge process and lab scale
experiments (Layton et al., 2000; Ternes et al., 1999a). However, other investigations
indicated only a partially elimination of estrogens, depending on facility and location of the
respective wastewater treatment plant and residual amounts could reach surface and
groundwater (Ternes et al., 1999b; Desbrow et al., 1998; Belfroid et al., 1999). Layton et al.
(2000) have performed a series of biodegradation studies with radio-labeled estradiol and
testosterone in laboratory assays inoculated with activated sludge obtained from different
wastewater treatment plants. Differences in mineralization of estradiol by sludge from a
municipal compared to that from an industrial plant was observed. In contrast to estradiol,
testosterone was mineralized to carbon dioxide in all investigated plants.
4